Antonio De Marco

Digital filtering with a sinusoidal window function: An alternative technique for resolution enhancement in FT NMR

Abstract As an alternative to convolution difference techniques for resolution enhancement in the 1 H NMR spectra of proteins, digital filtering of the FID with a sinusoidal window function is suggested. As an illustration, this “sine bell routine” was applied to the high-field region of the 1 H NMR spectrum at 360 MHz of the basic pancreatic trypsin inhibitor.

1H NMR studies at 360 Mhz of the methyl groups in native and chemically modified basic pancreatic trypsin inhibitor (BPTI)

In the 1H NMR spectra obtained at 360 MHz after digital resolution enhancement, the multiplet resonances of the methyl groups in the basic pancreatic trypsin inhibitor (BPTI) were resolved. With suitable double irradiation techniques the individual methyl resonances were assigned to the different types of aliphatic amino acid residues. Furthermore, from pH titration and comparison of the native protein with chemically modified BPTI, the resonance lines of Ala 16 in the active site and Ala 58 at the C-terminus were identified. Potential applications of the resolved methyl resonances as natural NMR probes for studies of the molecular conformations are discussed.

Isolation and characterization of a new form of the porcine pancreatic secretory trypsin inhibitor Biochemical studies and high-resulution 1H-NMR

Abstract A new active form of porcine PSTI (pancreatic secretory trypsin inhibitor) was isolated during the fractionation by ion-exchange chromatography of the already known forms PSTI I and II. Biochemical and 1H-NMR techniques were used to characterize the new inhibitor, which is referred to as PSTI III. The amino acid composition, the nature of the N-terminal residue and data obtained from the tryptic peptides all indicate that PSTI III lacks the N-terminal octapeptide of PSTI I; hence, it starts and ends with disulfide bridges. The conclusion is supported by the 1H-NMR spectrum of the protein at 270 MHz. The biological activity and the most prominent conformational and dynamic features of forms I and II are retained in inhibitor IIL However, PSTI III appears to be less compact than its parent forms I and II, suggesting that in the latter inhibitors an interaction between the N-terminal tail and the bulk of the protein may contribute to the overall stability. The genetic origin of PSTI III is discussed.

Epidermal growth factor: exposure and dynamics of the aromatic side chains as investigated by photo-CIDNP and variable temperature 1H-NMR

EGF is a 53 residue polypeptide of multiple biological activities. Homonuclear decoupling, spin‐echo multiplet selection and Photo‐CIDNP experiments lead us to fully assign the resonances from the aromatic side chains (5 Tyr, 1 His and 2 Trp). The photochemical experiment gives specific attribution of doublets in tyrosines and multiplets in tryptophans. The resonances from two tyrosines are broadened and shifted at high fields, suggesting the existence of hydrophobic domains in EGF, consistent with the presence of ring current shifted methyl resonances. However, the amide exchange in D2O solution is considerably faster than that observed for globular proteins of the same size, and most of the aromatic residues are accessible to the flavin dye. The combined evidence suggests that EGF possesses a folded structure, but the molecule is rather floppy. From spectra measured at variable temperature a ΔG° at 25°C of about 8 kcal/mol is calculated.

1H-NMR of reverse micelles. II: Conformational studies of peptides and proteins in the AOT/water/isooctane system

The interaction of AOT reverse micelles with Met-enkephalin, the pancreatic secretory trypsin inhibitor (PSTI) and the epidermal growth factor (EGF) is examined by NMR methods and the three systems are compared. While Met-enkephalin adopts a folded conformation, PSTI appears to become highly flexible, suggestive of a non-specific interaction with the micelles. On the other hand, the EGF spectrum shows that, although the main globular features of the protein are retained in the presence of AOT, the C-terminal fragment has to rearrange its conformation when put in contact with the micelle wall.

1H-NMR of reverse micelles. I: The surfactant resonances as probes for the AOT/water/isooctane system

1H-NMR spectra of Aerosol-OT (AOT) reverse micelles in isooctane are reported at various water contents and several temperatures. The resonances from the AOT protons near the polar head are completely assigned. The NMR parameters (chemical shift and linewidth) appear to be suitable probes for characterizing the physico-chemical state of micelles, in particular in order to define the range in which the system is fully homogeneous and transparent. The results correlate well with those obtained from optical density measurements. Lanthanide ions dissolved in the water phase selectively perturb the AOT resonances from the protons nearest to the polar head; conversely, non-polar shift reagents soluble in the organic phase do not cause appreciable effects on any of the observable signals.

Conformation of a hexahydrocannabinol derivative via 1- and 2-D noe experiments. evaluation of 1JCH coupling constants by semiselective 2-D J-spectroscopy and ther correlation with the ring geometry

Abstract The conformation of the hexahydrocannabinol derivative (-)-(6aR,9R,10aR)-6, 6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-dibenzo[ b , d ]pyran (HHC) has been determined by 1- and 2-D NOE experiments, the latter obtained with a pulse sequence which strongly reduces the contribution of the main diagonal. 1JCH and 3JCH have been measured in heteronuclear J-resolved 2-D spectra obtained both with the gated decoupling technique, and with “spinflip” of directly attached protons (“semlselective” 2-D J-spectroscopy). On the basis of the NOE effects, it can be concluded that the conformation of the pyran ring is essentially half-chair. This result is consistent with the 3JCH measured between the geminal methyl carbons at C-6 and H-6a; at least one of the two couplings appears to be sensibly lowered by the nearby oxygen. The 1JCHs appear to be directly related to the proton geometry, e.g. they are 127.7 Hz for the three equatorial protons at sites 7, 8 and 10 (see Fig. 1), and range between 123.4 and 124.8 Hz for the corresponding axial protons.